Antigen binding proteins involved in the immune response are present in mammals as large polyclonal repertoires representing a broad diversity of binding specificities. This diversity is generated by rearrangement of gene sequences encoding variable regions of these binding proteins. Such variable region binding proteins include soluble and membrane-bound forms of the B cell receptor (also known as immunoglobulins or antibodies) and the membrane-bound T cell receptors (TcR). With respect to immunoglobulins, their affinity is enhanced subsequent to recognition of an antigen by a B cell antigen receptor, through a process termed affinity maturation which involves cycles of somatic hypermutation of these variable genes.
Notably, immunoglobulins or fragments thereof, such as Fab fragments, Fv fragments and single chain Fv (scFv) molecules have been subject to cloning and recombinant expression. However, all other variable region binding proteins can in principle also be cloned and expressed using the same concepts as for antibodies.
Known approaches for isolating antibodies with a desired binding specificity most often involves generation of hybridomas from immunized hosts followed by screening for specific clones or involves the generation of combinatorial expression libraries in E. coli composed of immunoglobulin variable domains, which are subsequently enriched using techniques such as, for example, phage display.
The main restriction in the use of the hybridoma technology for making therapeutic antibodies is the absence of a human lymphoma suitable as fusion partners for human B lymphocytes. Heterohybridomas (i.e., fusion of human B cells with mouse lymphomas) are notoriously unstable and thus rarely lead to suitable cell lines for production purposes. Human B cells immortalized through infection with Epstein-Barr virus exhibit similar challenges of instability. The lack of robust cellular methodology for making human antibodies for therapy can be compensated with more recent advantages in molecular biology.
The use of combinatorial libraries and phage display allows for generation of large repertoires of antibody clones with a potential diversity in excess of 1010. From this repertoire selection for binding to a specific target can be performed thereby generating a sub-library. This sub-library can be used to generate either polyclonal or monoclonal antibodies. The variable region encoding sequences (for example immunoglobulin heavy chain variable region and light chain variable region encoding sequences) which constitute the library can be amplified from lymphocytes, plasma cells, hybridomas or any other immunoglobulin expressing population of cells. Current technologies for generating combinatorial libraries involve separate isolation of the variable region encoding sequences from a population of cells. Thus, the original pairing of for example immunoglobulin heavy chain variable region and light chain variable region encoding sequences will be lost. Rather, in a combinatorial library said sequences are randomly paired and the original combinations of these variable sequences will only occur by chance. Thus, in order to isolate variable region encoding sequences responsible for a desired binding specificity, a considerable amount of screening is necessary. This is typically performed in combination with methods for enrichment of clones exhibiting a desired specificity, such as ribosome display or phage display. Even then, the diversity achieved might not be sufficiently large to isolate variable region encoding sequence pairs giving rise to binding proteins of similar high affinity as those found in the original cells. Further, the enrichment procedures normally used to screen combinatorial libraries introduce a strong bias e.g. for polypeptides of particular low toxicity in E. coli, efficient folding, slow off-rates, or other system dependent parameters, that reduce the diversity of the library even further. In addition, clones derived from such combinatorial libraries will be more prone to produce binding proteins with cross-reactivity against self-antigens because they as pairs, in contrast to original pairs (hereafter called cognate pairs), never have been through in vivo negative selection against self-antigens, such as it is the case for B and T lymphocyte receptors during particular stages of their development. Therefore, the cloning of original pairs of variable region encoding sequences is a desirable approach. Moreover, the frequency of clones exhibiting a desired binding specificity is expected to be considerably higher within a library of cognate pairs, than in a conventional combinatorial library, particularly if the starting material cells are derived from a donor with high frequency of cells encoding specific binding pairs e.g. immune or immunized donors. It follows that the size of a cognate pair library will not need to be as large as a combinatorial library: a cognate pair library size of 104 to 105 clones or even as small as 102 to 103 clones derived from a donor with a relevant ongoing immune response might very well suffice in order to obtain binding proteins representing a broad diversity of desired binding specificities.
In order to generate cognate pair libraries the linkage of the variable region encoding sequences derived from the same cell is required. At present, two different approaches which can achieve cognate pairing of variable region encoding sequences have been described.
In-cell PCR is an approach where a population of cells is fixed and permeabilized, followed by in-cell linkage of heavy chain variable region and light chain variable region encoding sequences from immunoglobulins. This linkage can be performed either by overlap-extension RT-PCR (WO 93/03151) or by recombination (Chapal, N. et al. 1997 BioTechniques 23, 518-524). The amplification process as described in these publications is a three or four step process consisting of i) reverse transcription utilizing constant region primers generating immunoglobulin cDNA, ii) PCR amplification of the heavy and light chain variable region encoding sequences utilizing primer sets containing either overlap-extension design or recombination sites, iii) linkage by recombination, if this approach is chosen, iv) nested PCR of the products generating restriction sites for cloning. Since the cells are permeabilized there is a considerable risk that amplification products might leak out of the cells, thereby introducing scrambling of the heavy chain variable region and light chain variable region encoding sequences, resulting in the loss of cognate pairing. Therefore, the procedure includes washing steps after each reaction which makes the process laborious and reduces the efficiency of the reactions.
More generally, the in-cell PCR is notoriously inefficient, resulting in low sensitivity. Accordingly, the in-cell PCR linkage technique has never found widespread usage, and the original study has in fact never been reliably repeated in a way which can be used to verify that the linkage actually occurs within the cell. This, however, is absolutely crucial to avoid scrambling of the heavy chain variable region and light chain variable region encoding sequences and thereby disrupting the cognate pairs.
A different in-cell approach is described in WO 01/92291. This approach is based on RNA trans-splicing, and achieves joining of VH and VL encoding mRNA within the cell. This approach requires the presence of a DNA construct driving the trans-splicing within the cells.
Single-cell PCR is a different approach to achieve cognate pairing of heavy chain variable region and light chain variable region encoding sequences (see, for example, Coronella, J. A. et al. 2000 Nucleic Acids Res. 28, E85; Wang, X., et al. 2000 J. Immunol. Methods 20, 217-225). In these publications a population of immunoglobulin expressing cells are distributed by diluting to a density of one cell per reaction, thereby eliminating scrambling of heavy chain variable region and light chain variable region encoding sequences during the cloning process. Basically, the process described is a three to four step procedure consisting of i) reverse transcription utilizing oligo-dT-, random hexamer- or constant region primers generating cDNA, ii) fractionating the cDNA product into several tubes and performing PCR amplification on the individual variable chain encoding sequences (in separate tubes) with primer sets containing restriction sites for cloning, iii) nested PCR of the products generating restriction sites for cloning (optionally) and iv) linking the heavy chain variable region and light chain variable region encoding sequences from the separate tubes by cloning them into an appropriate vector, which in itself is a multi-step process.
In humans there are two types of light chains: lambda (λ) and kappa (κ). This means that with the cDNA generated from every single cell at least three separate PCR reactions must be performed followed by analysis and cloning of the appropriate fragments into a single vector to achieve the cognate pairing. Thus, the single-cell PCR approach as described requires a large number of manipulations to generate a library of cognate pairs. Although, a cognate pair library does not need to be as large as a combinatorial library in order to obtain binding proteins representing a broad diversity of binding specificities it would still be a laborious task to generate a library of for example 104 to 105 clones by the described single-cell PCR approach. Further, the large number of manipulations highly increases the risk of contamination and human error.
In order to obtain high affinity binding proteins corresponding to the affinities normally observed during an immune response, cognate pairing of the variable region sequences in association with their amplification is highly advantageous. To generate a library of large diversity it is necessary to have a cloning technique that can be fitted to a high-throughput format and where the risk of contamination and scrambling is minimal.
A reduction of the number of cloning steps allowing the generation of combinatorial libraries to be fitted to a high-throughput format, is likewise desired.